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| United States Patent |
5,712,120
|
|
Rodriguez
, et al.
|
January 27, 1998
|
Method for obtaining modified immunoglobulins with reduced
immunogenicity of murine antibody variable domains, compositions
containing them
Abstract
Modified chimaeric antibodies, and antibody heavy and light chains, which
comprise variable domains derived from a first mammalian species, usually
mouse, and constant domains from a second mammalian species, usually
human. Modification concerns the variable domains, in particular the
framework regions of the variable domains. The modifications are made only
in T-cell antigenic structures present in framework regions, and do not
cover canonical structures or Vernier zone. The modifications adapt the
amino acid sequences concerned to those occurring in corresponding
antibodies derived from said second mammalian species. Thus, the modified
chimaeric antibodies retain the original antigen recognition and binding
properties but become less immunogenic to said second mammalian species,
which improves their therapeutical utility with said second mammalian
species. Recombinant DNA technology may be used to construct and produce
the modified chimaeric antibodies.
| Inventors:
|
Rodriguez; Rolando Perez (Vibora, CU);
Mateo de Acosta del Rio; Christina Maria (Vedado, CU);
Valladares; Josefa Lombardero (Vibora, CU)
|
| Assignee:
|
Centro De Immunologia Molecular (Havana, CU)
|
| Appl. No.:
|
497312 |
| Filed:
|
June 30, 1995 |
Foreign Application Priority Data
| Current U.S. Class: |
435/69.6; 424/133.1; 435/69.7; 435/70.21; 435/71.1; 435/328; 530/387.3; 536/23.53 |
| Intern'l Class: |
C12N 015/13; C07K 016/00; C07H 021/04; A61K 039/395 |
| Field of Search: |
435/69.6,172.3,70.21,71.1,328,69.7
424/133.1,135.1
530/387.3
536/23.53
|
References Cited
| Foreign Patent Documents |
| 0 519 596 A1 | Dec., 1992 | EP.
| |
Other References
Article entitled: A Possible Procedure for Reducing the Immunogenicity of
Antibody Variable Domains While Preserving Their Ligand-Binding
Properties. Author: Eduardo A. Padlan Published in Molecular Immunology,
vol. 28, No. 4/5, pp. 489-498, 1991.
Reyes, VE et al. Methods in Enzymology 202:225-238 (1991).
Margalit, H. et al. Journal of Immunology 138(7):2213-2229 (1987).
Foote J. et al Journal of Molecular Biology 224:487-499 (1992).
Chothia C. et al. Journal of Molecular Biology 196:901-917 (1987).
|
Primary Examiner: Scheiner; Toni R.
Assistant Examiner: Lucas; John
Attorney, Agent or Firm: Trask, Britt & Rossa
Claims
We claim:
1. A method of modifying an antibody comprising:
comparing the framework amino acids of a variable domain of a first
mammalian species with a group of framework amino acid residue variable
domains of a second mammalian species;
determining a subgroup of the second mammalian species to which the first
mammalian species most closely corresponds;
selecting the antibody from said subgroup whose framework is most similar
to the first mammalian species' framework sequence;
identifying amino acid residues of the first mammalian species which differ
from the amino acid residues of the selected second mammalian species
framework and which are within T-cell antigenic sequences, with said amino
acid residues being with T-cell antigenic sequences in the variable region
of the immunoglobulins;
identifying only those amino acid residues which are not within a
complementarity region or are not directly involved with canonical
structures or Vernier zone; and
replacing the amino acid residues in the first mammalian species framework
which differ from the amino acid residues of the second mammalian species
with the corresponding amino acid residues from the most similar second
mammalian species thus identified; and
obtaining the modified antibody.
2. The method of claim 1 wherein the first mammalian species is mouse.
3. The method of claim 1 wherein the second mammalian species is human.
4. The method of claim 1, wherein one or more heavy chain constant domains,
the light chain constant domain, or both heavy and light chain constant
domains of said first mammalian species antibody are replaced by the
corresponding constant domain of the second mammalian species antibody.
Description
FIELD OF THE INVENTION
The present invention is related to the field of immunology, in particular
to a method for obtaining modified immunoglobulins with reduced
immunogenicity of murine antibody variable domains and compositions
containing them.
BACKGROUND OF THE INVENTION
The immune system builds antibodies that bind to a vast range of antigens
with high avidity and specificity, and trigger effector mechanisms.
Antibodies have been used in medicine as diagnostic and therapeutic
agents, and their potential has been successively enhanced with the advent
of new technologies.
Hybridoma technology allowed isolation of cell lines secreting antibodies
of a single specificity (Kohler G., Milsrein C. (1975) Nature (London)
256, 495-497), and gene technology has allowed the construction of a range
of engineered antibodies from hybridomas.
Engineering of antibodies is facilitated by their domain structure and may
further improve the utility of many antibodies by the acquisition or loss
of some of their properties. The antigen-binding properties of the
antibody provide the recognition function and this can be combined with to
one or more of a number of effector agents. The combination of these two
features must then be tested against the criteria of efficacy, specificity
and immunogenicity.
Monoclonal antibody producing hybridomas have been most readily obtained
from immunized rodents. At present, the use of several murine monoclonal
antibodies has been widespreaded for the imaging and treatment of
malignancy, prophylactic administration to guard against toxic shock,
modification of graft rejection episodes, and to temper acute inflammatory
reactions.
In most of the cases where rodent antibodies have been used for therapy,
the recipients have elicited an immune response directed towards the
antibodies themselves. Such reactions have limited the duration and
effectiveness of the therapy.
Development of similar reagents from human sources has been frustated,
although several options exist, using for example SCID-hu mice, in vitro
immunization, recombinatorial libraries, or some useful combination of
these. Because there are many well-characterized rodent monoclonal
antibodies already available which might be used in the clinic if the
immune response could be abolished, the production of engineered
antibodies has received much attention.
Engineered antibodies have been designed to replace as much as possible of
the xenogeneic sequences with the equivalent human sequence. Among the
genetically engineered antibodies are chimaeric antibodies in which
segments from immunoglobulins from diverse species are joined together.
Initially, chimaeric antibodies were constructed containing the rodent
variable regions fused to human constant domains. Particularly mouse/human
chimaeric antibodies are potentially useful for immunotherapy for they
should exhibit the same specificity but reduced immunogenicity compared to
their murine counterparts. The following references describe chimaeric
antibody technology: Lobuglio et al, Proc. Natl. Acad. Sci. USA 86:
4220-4224 (1989); U.S. Pat. No. 4,816,567; PCT International Publication
No. WO 87/02671 published May 7, 1987; European Patent Publication No.
255,694 published Feb. 10, 1988; European Patent Publication No. 274,394
published Jul. 13, 1988; European Patent Publication No. 323,806 published
Jul. 12, 1989; PCT International Publication No. WO 89/00999 published
Feb. 9, 1989; European Patent Publication No. 327,000 published Aug. 9,
1989; European Patent Publication No. 328,404 published Aug. 16, 1989; and
European patent Publication No. 332,424 published Sep. 12, 1989.
It is worth noting that even the replacement of the Constant regions with
human equivalents may not effectively reduce their immunogenicity. Still
approximately half of the recipients mounted an immune response to the
rodent variable regions. Subsequently, rodent antibodies have been
extensively manipulated to resemble more fully human antibodies.
Further reduction in the immunogenicity of chimaeric antibodies has been
achieved by grafting only the complementarity determining regions (CDRs)
from the rodent monoclonal antibody onto human framework regions (FRs)
prior to its subsequent fusion with an appropriate constant domain (Jones
et al, Nature 321: 522-525 (1986)). This procedure to accomplish
CDR-grafting often results in imperfectly humanized antibodies, for
example, the resultant antibody has either lost affinity or in an attempt
to retain its original affinity a number of the murine framework residues
have replaced the corresponding ones of the chosen human framework
(Winter, European Patent Application, Publication No. 239,400; Riechmann
et al, Nature 332: 323-327 (1988)).
Strategies have has been developed with the objective of identifying the
minimum number of residues for transfer to achieve a useful binding
affinity with the least potential consequences on immunogenicity. However,
it has emerged that each of these strategies has only been successful to
some degree in the reconstitution of parental affinity.
The ligand binding characteristics of an antibody combining site are
determined primarily by the structure and relative disposition of the
CDRs, although some neighbouring framework residues also have been found
to be involved in antigen binding (Davies et al, Ann. Rev. Biochem. 59:
439-473 (1990)). Thus, the fine specificity of an antibody can be
preserved if its CDR structures and some of the neighbouring residues,
their interaction with each other, and their interaction with the rest of
the variable domains can be strictly maintained.
A further procedure for the humanization of an antibody has been suggested
by Padlan (Padlan, European Patent Application, Publication No. 0 519 596
A1; Padlan, Molecular Immunology 28: 489-498 (1991)). It is based on the
fact that the antigenicity of a protein is dependent on the nature of its
surface, and a number of the solvent-accessible residues in the rodent
variable region are substituted by residues from a human antibody. The
locations of these residues are identified from an inspection of the high
resolution X-ray structures of the human antibody KOL and the murine
antibody J539. The choice of the human surface residues is arrived at by
identifying the most homologous antibody sub-group.
The nature of the protein surface is important for its recognition and
internalization by antigen-processing cells, specifically by
antigen-specific B-cells. In addition, the recognition of specific linear
sequences by T-cells is also an important element in the immunogenicity of
proteins.
Several groups have developed automated-computerized methods for the
identification of sequence features and structural determinants that play
a role in the MHC restriction of helper T-cell antigenic peptides
(Bersofsky et al, J. Immunol. 138: 2213-2229 (1987), Elliott et al, J.
Immunol. 138: 2949-2952 (1987), Reyes et al, J. Biol. Chem. 264:
12854-12858 (1989)). Using these algorithms, it has been possible to
identify predicted T cell-presented peptides.
Analysis of antibodies of known atomic structure has elucidated
relationships between the sequence and three-dimensional structure of
antibody combining sites (Chothia et al, J. Biol. Chem. 196: 901-917
(1987)). These relationships imply that, except for the third region in
the VH domains, binding site loops have one of a small number of
main-chain conformations: "Canonical structures". The canonical structure
formed in a particular loop is determined by its size and the presence of
certain residues at key sites in both the loop and in framework regions.
An additional subset of framework residues has been defined as a "Vernier"
zone, which may adjust CDR structure and fine-tune the fit to antigen
(Foot et al, J. Mol. Biol. 224: 487-499 (1992)). Substitutions of these
residues have been shown to be important to restoring the affinity in CDR
grafted antibodies, so the Vernier zone has an obvious consequence for the
design of humanized antibodies.
SUMMARY OF THE INVENTION
The invention provides a means of converting a monoclonal antibody of one
mammalian species to a monoclonal antibody of another species. The
invention is useful in predicting potential T-epitopes within the sequence
of variable regions. The invention is useful in identifying the amino acid
residues responsible for species specificity or immunogenicity within the
sequence of the monoclonal antibody responsible of the T-immunogenicity.
Another aspect of the invention is to judiciously replace the amino acid
residues within the T-epitope sequences of one species with those of a
second species so that the antibodies of the first species will not be
immunogenic in the second species. A further aspect involves providing
replacements only in the framework regions of the heavy and light chains
and not in the complementarity determining regions; also the amino acids
belonging to the Vernier zone and those involved in the canonical
structures cannot be replaced. Another aspect involves providing novel DNA
sequences incorporating the replacement amino acid residues. Another
aspect involves providing a vector containing the DNA sequences for the
altered antibody. Another aspect involves providing a eukaryotic or
procaryotic host transformed with a vector containing the DNA sequence for
the modified antibody.
A unique method is disclosed for identifying and replacing amino acid
residues within T-cell antigenic sequences which converts immunoglobulin
antigenicity of a first mammalian species to that of a second mammalian
species. The method will simultaneously change immunogenicity and strictly
preserve ligand binding properties. A judicious replacement of those amino
acid residues within T-cell antigenic sequences of the variable regions,
which are not involved in the three-dimensional structure, has no effect
on the ligand binding properties but greatly alters immunogenicity.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Deduced amino acid sequence of (a) VK and (b) VH of murine R3
antibody. CDRs are underlined.
FIGS. 2 and 3 Analysis for the modification of the variable regions of
heavy and light chains of antibody IOR-R3.
A: sequence of the variable region of the murine IOR-R3 monoclonal
antibody.
B: sequence of the variable region of the most homologous human
immunoglobulin.
C: sequence of the modified variable region of IOR-R3.
shading: predicted T-cell antigenic sequences.
underlined amino acid residues: amino acids involved in tertiary structure.
bold font: complementarity determining regions.
amino acid residues in boxes: proposed replacements.
The description is the same for both, heavy and light chains.
FIG. 4 Molecular model of the variable region of mAb R3 displayed as a
ribbon. VH is on the right and is darker than VL. The model shows the side
chain of murine residues that were mutated in order to humanize the
predicted amphipatic segments.
FIG. 5 Detection of binding of the chimaeric and mutant R3 to EGF-R by RRA.
Antigen binding activity was assayed in different concentrations of
purified murine R3 (-.-), chimaeric R3 (+) and mutant VHR3/muR3VK (*) and
plotted as CPM of bound .sup.125 I-EGF against log of the concentration of
each antibody. (concentration of IgG was quantitated by ELISA.)
FIG. 6 Immunization of monkeys with murine R3, chimaeric R3 and mutant R3.
ordinates: Absorbance at 405 nm.
abscises: number of days of blood collected.
The ELISA was performed as described in EXAMPLE 9. The arrows indicate the
time of intravenous injection of 2 mg of each mAb. The serum dilution used
was 1/10,000.
FIGS. 7 and 8 Analysis for the modification of the variable regions of
heavy and light chains of antibody IOR-T1.
A: sequence of the variable region of the murine IOR-T1 monoclonal
antibody.
B: sequence of the variable region of the most homologous human
immunoglobulin.
C: sequence of the modified variable region of IOR-T1 antibody.
The symbols are the same as in FIG. 2. The description is the same for
both, heavy and light chains.
FIGS. 9 and 10: Analysis for the modification of the variable regions of
heavy and light chains of antibody IOR-CEA1.
A: sequence of the variable region of the murine IOR-CEA1 monoclonal
antibody.
B: sequence of the variable region of the most homologous human
immunoglobulin.
C: sequence of the modified variable region of IOR-CEA1 antibody.
The symbols are the same as in FIG. 2. The description is the same for
both, heavy and light chains.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a procedure which simultaneously reduces
immunogenicity of the rodent monoclonal antibody while preserving its
ligand binding properties in its entirety. Since the antigenicity of an
immunoglobulin is dependent on the presence of T-cell antigenic peptides
within its sequence, the immunogenicity of a xenogenic or allogenic
antibody could be reduced by replacing the residues included in the T-cell
antigenic sequences which differ from those usually found in antibodies of
another mammalian species.
The replacement of residues does not include those involved in the
canonical structures or in the Vernier zone. This judicious replacement of
residues has no effect on the structural determinants or on the
interdomain contacts, thus, ligand binding properties should be unaffected
as a consequence of alterations which are limited to the variable region
framework residues.
(1) Analysis of Homology of Variable Regions
The present procedure makes use of the available sequence data for human
antibody variable domains compiled by Kabat et al, "Sequences of proteins
of Immunological Interest", Fifth edition, Bethesda, Md.; National Inst.
of Health, 1994.
In the first step the variable domains of any heavy or light chain of a
first animal species, e.g. the mouse, are compared with the corresponding
variable domains of a second animal species, e.g. human. It is intended
that this invention will allow the antigenic alteration of any animal
species antibody.
The comparison is made by an automated-computerized method (PC-DOS HIBIO
PROSIS 06-00, Hitachi). The most homologous human variable regions are
then compared, residue for residue, to the corresponding murine regions.
This will also define the human subgroup to which each mouse sequence most
closely resembles.
(2) Prediction of T-epitopes
In the second step, the two homologous variable region sequences, mouse and
human, are analysed for the prediction of T-antigenic sequences.
The algorithm AMPHI (Bersofsky et al, The Journal of Immunology 138:
2213-2229 (1987)) predicts .alpha. Helical sequences. The algorithm SOHHA
predicts the strip of helix hydrophobicity (Elliott et al, J. Immunol.
138: 2949-2952 (1987)). These algorithms predict T-cell presented
fragments of antigenic proteins.
(3) Analysis for Immunogenicity Reduction
Those residues in the mouse framework which differ from its human
counterpart are replaced by the residues present in the human counterpart.
This switching (replacement) occurs only with those residues which are in
the T-antigenic sequences.
Finally, replacement of those residues responsible for the canonical
structures or those involved in the Vernier zone could have a significant
effect on the tertiary structure. Hence, they cannot be included in the
replacement. Additional information about the influence of the proposed
replacements on tertiary structure or the binding site could be obtained
from a molecular model of the variable regions.
The molecular model can be built on a Silicon Graphics Iris 4D workstation
running UNIX and using the molecular modeling package "QUANTA" (Polygen
Corp.).
(4) Method for Constructing and Expressing the Altered Antibody
The following procedures are used to prepare recombinant DNA sequences
which incorporate the CDRs of a first mammalian species, usually animal,
e.g. murine mAb, both light and heavy chains, into a second mammalian
species, preferably human, appearing frameworks that can be used to
transfect mammalian cells for the expression of recombinant antibody less
immunogenic and with the antigen specificity of the animal monoclonal
antibody.
The present invention further comprises a method for constructing and
expressing the modified antibody comprising:
a.-) mutagenesis and assembly of variable region domains including CDRs and
FRs regions. The PCR-mutagenesis method (Kamman et al, Nucleic Acids Res.
17: 5404-5409 (1989)) is preferably used to introduce the changes at
different positions.
b.-) preparation of an expression vector including one variable region and
the corresponding human constant region which upon transfection into cells
results in the secretion of protein sufficient for affinity and
specificity determinations.
c.-) co-transfection of heavy and light chain expression vectors in
appropriate cell lines.
After about 2 weeks, the cell supernatants are analyzed by ELISA for human
IgG production. The samples are then analysed by any method for human IgG
capable of binding to specific antigens.
The present invention provides a method for incorporating CDRs from animal
monoclonal antibodies into frameworks which appear to be human
immunoglobulin in nature so that the resulting recombinant antibody will
be either weakly immunogenic or non-immunogenic when administered to
humans. Preferably, the recombinant immunoglobulins will be recognized as
self proteins when administered for therapeutic purpose. This method will
render the recombinant antibodies useful as therapeutic agents because
they will be either weakly immunogenic or non-immunogenic when
administered to humans.
The invention is further contemplated to include the recombinant conversion
of any animal monoclonal antibody into a recombinant human-appearing
monoclonal antibody by providing that with a suitable framework region.
The invention is intended to include the conversion of any animal
immunoglobulin to a human-appearing immunoglobulin. It is further intended
that human-appearing immunoglobulin can contain either Kappa or Lambda
light chains or be one of any of the following heavy chain isotypes
(alpha, delta, epsilon, gamma and mu).
The following examples intend to ilustrate the invention but not to limit
the scope of the invention.
EXAMPLE 1
Murine Variable Region of R3 Monoclonal Antibody DNA Sequencing
Cytoplasmic RNA was extracted from about 10.sup.6 R3 (anti Epidermal growth
Factor receptor) hybridoma cells as described by Faloro et al (Faloro, J.
et al, Methods in Enzymology 65: 718-749, 1989).
The cDNA synthesis reaction consisted of 5 ug RNA, 50 mM Tris-HCl, pH 7.5,
75 mM KCl, 10 mM DTT, 3 mM MgCl.sub.2, 25 pmol of CG2AFOR primer (5'
GGAAGCTTAGACCGATGGGGCCTGTTGTTTTG 3') for the heavy chain variable region
or CK2FOR primer (5' GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3') for the light
chain variable region, 250 uM each of dATP, dTTP, dCTP, dGTP, 15 U
ribonuclease inhibitor (RNA guard, Pharmacia) in a total volume of 50 ul.
Samples were heated at 70.degree. C. for 10 min and slowly cooled to
37.degree. C. over a period of 30 min. Then, 100 units MMLV reverse
transcriptase (BRL) were added and the incubation at 37.degree. C.
continued for 1 hour.
The VH and VK cDNAs were amplified using the PCR as described by Orlandi et
al (Orlandi, R. et al, Proc. Natl. Acad. Sci. USA 86: 3833-3837, 1989).
For PCR amplification of VH, DNA/primer mixtures consisted of 5 ul cDNA,
25 pmoles of CG2AFOR primer (5' GGAAGCTTAGACCGATGGGGCCTGTTGTTTTG 3') and
VH1BACK primer (5' AGGT(G/C)(A/C)A(A/G)CTGCAG(G/C)AGTC(A/T)GG 3').
For PCR amplification of VK, DNA/primers mixtures consisted of 5 ul cDNA
and 25 pmoles of CK2FOR primer (5' GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3')
and VK10BACK primer (5' TTGAATTCCAGTGATGTTTTGATGACCCA 3'). To these
mixtures were added 2.5 mM each of dATP, dCTP, dTTP, and dGTP, 5 ul
constituents of 10X buffer thermolase and 1 unit of Thermolase (IBI) in a
final volume of 50 ul. Samples were subjected to 25 thermal cycles at
94.degree. C., 30 sec; 50.degree. C., 30 sec; 72.degree. C., 1 min; and a
last incubation for 5 min at 72.degree. C. Amplified VH and VK DNA were
purified on Prep. A Gene purification kit (BioRad).
The purified VH and VK cDNA were cloned into M13 vector. Clones were
sequenced by the dideoxy method using T7 DNA Pol (Pharmacia). See FIG. 1.
EXAMPLE 2
Construction of Chimaeric Genes
We reamplified the cDNA by PCR using VH1BACK primer (5'
AGGT(G/C)(A/C)A(A/G)CTGCAG(G/C)AGTC(A/T)GG 3') and VH1FOR primer (5'
TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG 3') for VH and VK3BACK primer
(5'GACATTCAGCTGACCCA 3') and VK3FOR primer (5' GTTAGATCTCCAGTTTGGTGCT 3')
for VK. The amplified cDNAS were digested with PstI and BstEII for the VH
gene or PvuII and BglII for the VK gene. The fragments were cloned into
M13-VHPCR1 (digested with PstI and BstEII) or into M13-VKPCR1 (digested
with PvuII and BclI). Details of vectors are given by Orlandi, R. et al,
Proc. Natl. Acad. Sci. USA 86: 3833-3837, 1989. The M13VHPCR-R3 and
M13VKPCR-R3 containing V gene inserts were identified directly by
sequencing.
The VH gene together with the Ig heavy chain promoter, appropriate splicing
sites and signal peptide sequences were excised from M13 vectors by
digestion with HindIII and Bam HI and cloned into an expression vector
(pSVgpt). A human IgG1 constant region (Takahashi, N. et al, Cell 29:
718-749, 1982) was then added as a BamHI fragment. The resultant
construction was R3VH-pSVgpt. The construction of the R3VK-pSVhyg was
essentially the same except that the gpt gene was replaced by the
hygromicin resistance gene and a human Kappa chain constant region was
added (Hieter, P. A. et al, Cell 22: 197-207, 1980).
EXAMPLE 3
Modification of the Variable Domain Sequences of IOR-R3 Murine Monoclonal
Antibody to Humanize the Predicted T-cell Antigenic Sequences
The variable region sequences of heavy and light chains of R3 were analyzed
for T-cell antigenic sequences. It was made by using the computer
algorithm AMPHI, which predicts segments of the sequences 11 amino acids
in length with an amphipatic helix structure, that is have one side
hydrophobic and one side hydrophilic which bind to MHC II molecules.
Within the variable domain sequence of the heavy chain were predicted 5
segments which are (using Kabat's numbering):
1. FR1 between amino acids 3-13.
2. FR1 between amino acids 8-20.
3. FR2 and CDR2 between amino acids 39-55.
4. FR3 between amino acids 74-84.
5. FR4 and CDR3 between amino acids 100c-110.
FIG. 2 shows the sequences corresponding to the heavy chain.
This murine sequence is compared with the immunoglobulin sequences included
in the GeneBank and EMBL database. The most homologous human variable
region sequence is determined and also the human subgroup to which the
murine sequence most closely resembles is defined. In this case the human
sequence found was a fetal immunoglobulin called HUMIGHVA, which variable
region has 75% of homology with the FR regions of the murine
immunoglobulin R3.
Both variable region sequences, human and murine are then compared, residue
for residue, and those residues in FR regions which are not involved in
the vernier zone or with the canonical structures are selected. Therefore
they could be changed by those residues at the same position within the
human sequence.
Finally, this analysis is enriched with computer modeling of the binding
site. On the molecular model it is possible to define those replacements
which will perturb the tertiary structure of the binding site.
For the heavy chain of murine R3 we propose 6 replacements:
1. LEU at position 11 by VAL
2. VAL at position 12 by LYS
With only these two replacements it is possible to disrupt the amphipatic
helix and therefore the predicted T-epitope in the FR1.
3. SER at position 75 by THR
4. THR at position 76 by SER
5. ALA at position 78 by VAL
6. THR at position 83 by ARG
In this case, with the replacements proposed in the FR3, it is humanized.
The T-cell antigenic sequence in the FR2 contains two PRO which is a very
rare amino acid residue in most of the helical antigenic sites, so we
propose that it is not a real T-cell epitope.
In the position 108 at the FR4 appears THR which is present in the same
position in some human immunoglobulins, only residue 109 (LEU) is very
rare in human, except for this point difference most of the predicted
T-cell epitope is human, on this basis it does not need to be modified.
In FIG. 3 the analysis for the light chain of murine R3 is shown.
In the sequence only one amphipatic helix was predicted, between residue
52-63 corresponding to CDR2 and FR3, and in this region only one point
difference exists between murine and human sequences, at position 63. No
replacement is proposed, because this murine light chain should be
non-immunogenic in human (see molecular modelling).
EXAMPLE 4
Molecular Modelling of mAb R3 VK and VH
A model of the variable regions of mouse mAb R3 was built using the
molecular modeling program QUANTA/CHARm 4.0 (Molecular Simulations Inc.,
1994), running on a 150 MHz Silicon Graphics Indigo Extreme workstation.
The VK and VH frameworks were built separately from Fab 26-10 (Jeffrey, P.
D et al, Proc. Natl. Acad. Sci. USA 90, 10310, 1993) and Fab 36-71
(Strong, R. K. et al, Biochemistry 30, 3739, 1993), respectively. Fab
26-10 and mAb R3 have 92% homology in the VK frameworks and 88% homology
in the whole VK region. The VH frameworks of Fab 36-71 and mAb R3 have 85%
homology.
Coordinates were taken from the Brookhaven Protein Data Bank (entries IIGI
and 6FAB). The frameworks of Fab 36-71 were fitted to the frameworks of
Fab 26-10, matching only those residues that have been found to be often
involved in the interface between the light and heavy variable regions
(Chotia, C. et al, J. Mol. Biol. 186, 651, 1985). The VH domain of Fab
26-10 and the VK domain of Fab 36-71 were then deleted leaving the needed
hybrid. Side-chain replacements were performed following the maximum
overlap procedure (Snow, M. E. et al, Proteins 1, 267, 1986) and
comparing, where possible, with other crystal structures.
The hypervariable regions of the R3-Variable Light (VL) domain (L1, L2 and
L3) were built retaining the same main-chain conformations as in Fab
26-10, since the corresponding CDRs in both antibodies are highly
homologous and belong to the same canonical structural groups (Chotia, C.
et al, Nature 342, 877, 1989). In the VH domain of mAb R3, CDR HI belongs
to canonical structural group 1, as in Fab 36-71, so the main-chain
torsion angles of the parent molecule were kept. CDR H2 corresponds to
canonical structural group 2 and the main-chain conformation for this loop
was taken from the Fv fragment 4D5 (entry 1FVC), which was selected among
other highly resolved structures because of the good matching of its H2
loop base with the framework of Fab 36-71. For all the above mentioned
loops comparisons with other CDRs from the Data Bank were made to orient
the side chains.
To model CDR H3, which in mAiD R3 was 14 amino acids long, a high
temperature molecular dynamics was used for conformational sampling
(Bruccoleri, R. E. et al, Biopolymers 29, 1847, 1990). First, the whole
structure without CDR H3 was subjected to an energy minimization keeping
residues H-94 and H-103 fixed and using harmonic constraints of 10
Kcal/(mole atom A.sup.2) for main chain atoms. Then a loop was constructed
with an arbitrary conformation starting from the two previously fixed
amino acids. Those residues close to the framework were placed taking into
consideration other crystal structures and the top part of the loop was
built with an extended conformation avoiding strong steric interactions
with the rest of the molecule. For the next modeling steps only CDR H3 and
the neighbouring side chains within a distance of 5A.sup.0 were permitted
to move. An energy minimization was first carried out and then a molecular
dynamics at 800K was run for 150 picoseconds. The time step for the run
was set to 0.001 picosecond and coordinates were saved every 100 steps.
The 120 lowest energy conformations from the dynamics run were extracted
and subjected to an energy minimization in which all atoms in the
structure were allowed to move. Several low-energy conformations were
obtained and the one with the lowest energy was used in the subsequent
analyses. Differences between murine and humanized variants of R3 antibody
were individually modeled to investigate their possible influence on CDR
conformation.
Amino acid replacements in positions 11, 12 (FR1) and 83 (FR3) in the heavy
chain variable region are quite enough distant from the CDRs-FRs
boundaries and should not have any influence on binding affinity. SER 75
residue is pointing to outside, thus the replacement by THR seems not to
be important for binding capacity. By contrary THR 76 is accessible from
the top of the molecule and could be involved in the interaction with the
antigen. But the substitution of THR 76 by SER is a conservative change,
leading to no major variations in binding affinity probably.
The replacement of ALA 78 by VAL should not require steric rearrangements.
However VAL 78 could "push" forward ILE 34 (H|). In general, the proposed
point mutations should not affect binding affinity according to the
computer-aided molecular modelling study (FIG. 4).
The same analysis was done in the light chain variable region of IOR-R3,
molecular modelling indicates it is not necessary to make any changes in
this region.
EXAMPLE 5
Construction of Mutant Heavy Chain Variable Region of R3 by PCR Mutagenesis
The changes in the amino acids of mutant heavy chain variable region were
constructed using PCR mutagenesis (Kammann, M. et al, Proc. Natl. Acad.
Sci. USA 86, 4220-4224, 1989).
Briefly: Two amplification by PCR: the reaction mixture was: 0.5 ul the VH
supernatant of single strand DNA cloned in M13, 25 pmoles mutagenic oligo
1 or 2, 25 pmoles mutagenic oligo 3 or 4 primers (See below the primers
sequences). To these mixtures were added 2.5 mM each of dATP, dCTP, dTTP,
and dGTP, 5 ul constituents of 10X Vent Polymerase buffer (NEB) and 1 unit
of Vent DNA Polymerase (NEB) in a final volume of 50 ul. Samples were
subjected to 12-15 thermal cycles at 94.degree. C., 30 sec; 50.degree. C.,
30 sec; 75.degree. C., 1 min; and a last incubation for 5 min at
75.degree. C. The products of both PCRs are joined in a second PCR using
the outside primers only (3 and 4). Amplified VH DNA was purified on Prep.
A Gene purification kit (BioRad).
For the changes in the FR1 of LEU 11 and VAL 12 by VAL and LYS,
respectively, the following primers were used:
Primer 1: 5' GAAGCCCCAGGCTTCTTCACTTCAGCCCCAGGCTG 3'.
Primer 3: 5' GTAAAACGACGGCCAGT 3'.
These primers are combined in one PCR.
Primer 2: 5' CAGCCTGGGGCTGAAGTGAAGAAGCCTGGGGCTTCA 3'
Primer 4: 5' ACTGGCCGTCGTTTTAC 3'
These primers are combined in one PCR.
Then, the products of both PCRs are combined in one PCR using primers 3 and
4.
For the changes in the FR3, SER 75, THR 76, VAL 78 and THR 86 by THR, SER,
VAL and ARG, respectively, the following primers were designed:
Primer 1: 5'
GCAGAGTCCTCAGATCTCAGGCTGCTGAGTTGCATGTAGACTGTGCTGGTGGATTCGTCTACCGT 3'.
Primer 3: 5' GTAAAACGACGGCCAGT 3'.
These primers are combined in one PCR.
Primer 2: 5'
ACGGTAGACGAATCCACCAGCACAGTCTACATGCAACTCAGCAGCCTGAGATCTGAGGACTCTGC 3'
Primer 4: 5' ACTGGCCGTCGTTTTAC 3'.
These primers are combined in one PCR.
Then, the products of both PCRs are combined in one PCR using primers 3 and
4.
After mutagenesis VH genes were cloned in expression vectors (pSVgpt)
yielding the plasmids R3 mut VH-pSVgpt.
EXAMPLE 6
Transfection of DNA into NSO Cells
Four ug of R3VH-pSVgpt and 8 ug R3VK-pSVhyg (chimaeric) or R3 mutant
VH-pSVgpt and murine R3VK-pSVhyg were linearized by digestion with PvuI.
The DNAs were mixed together, ethanol precipitated and dissolved in 25 ul
water. Approximately 10.sup.7 NSO cells (Rat myeloma NSO is a non-Ig
secreting cell line) were grown to semiconfluency, harvested by
centrifugation and resuspended in 0.5 ml DMEN together with the digested
DNA in an electroporation single pulse of 170V at 960 uF (Gene-Pulser,
Bio-Rad) and left in ice for a further 30 min. The cells were then put
into 20 ml DMEN plus 10% fetal calf serum and allowed to recover for 24
hours. At this time the cells were distributed into a 96-well plate and
selective medium applied, transfected clones were visible with the naked
eyes 14 days later.
EXAMPLE 7
Quantification of IgG Production
The presence of human antibody in the medium of wells containing
transfected clones was measured by ELISA. Microtiter plate wells were
coated with goat anti-human IgG (heavy chain specific) antibodies
(Sera-Lab). After washing with PBST (phosphate buffered saline containing
0.02% Tween 20, pH 7.5), 20 ul of culture medium diluted in 100 ul of PBST
from the wells containing transfectants was added to each microtiter well
for 1 hour at 37.degree. C. The wells were then emptied, washed with PBST
and either peroxidase-conjugated goat anti human kappa (light chain
specific) region antibodies (Sera-Lab) were added and incubated at
37.degree. C. for 1 hour, the wells were then emptied, washed with PBST
and substrate buffer containing orthophenylenediamine added. Reactions
were stopped after a few minutes by the addition of sulphuric acid and
absorbance at 492 nm was measured.
EXAMPLE 8
EGF Receptor Radioligand Competition Assays
The determination of the affinity constant of the .sup.125 I-EGF binding to
its receptor by murine R3, chimaeric and mutant by rupture of epitopes T
antibodies was performed by a homogeneous Radio Receptor Analysis (RRA)
with human placenta microsomal fraction (Macias, A. et al, Interferony
Biotecnologia 2: 115-127, 1985).
These chimaeric and mutant by rupture of epitopes T antibodies were assayed
using this technique for its ability to bind to EGF-R (FIG. 5). Both
antibodies bound to EGF-R with the same affinity as the original murine
antibody (10.sup.-9 M), confirming that the correct mouse variable regions
had been cloned and the new antibody isotype did not affect binding. Even
more, the changes in the mutant antibody did not affect binding to the
antigen.
EXAMPLE 9
Immunization of Cercopithecus Aethiops Monkeys with the Murine, Chimaeric
and VH Mutant Antibodies
Three treatment groups with two Cercopithecus aethiops monkeys in each
group were immunized with murine R3 mAb, chimaeric R3 antibody and mutant
VH R3 antibody, respectively. All the groups were immunized subcutaneously
on days 0, 14, 28 and 42, with 2 mg of antibody adsorbed into 5 mg of
aluminum hydroxide.
Blood was collected prior to the first immunization and one week later of
each immunization, from all the groups, and the serum was obtained from
each sample, and kept at -20.degree. C. The titer of antibodies against
the murine R3 mAb was determined by an ELISA technique.
Costar plates (Inc, high binding) were coated with murine R3 monoclonal
antibody at a concentration of 10 ug/ml in bicarbonate buffer (pH 9.6) and
incubated overnight. Thereafter, the plates were washed with PBST, were
blocked with the same buffer containing 1% BSA during one hour at room
temperature.
The washing step was repeated and 50 ul/well of the different serum
dilutions were added. After incubating for 2 hours at 37.degree. C., the
plates were washed again and incubated 1 hour at 37.degree. C. with
alkaline phosphated conjugated goat anti-human total or anti-human IgG Fc
region specific antiserum (Sigma, Inc). After washing with PBST the wells
were incubated with 50 ul of substrate buffer (1 mg/ml of
p-nitrophenylphosphate diluted in diethanolamine buffer (pH 9.8)).
Absorbance at 405 nm in an ELISA reader (Organon Teknika, Inc).
A high IgG response to murine R3 antibody was obtained when this antibody
was used as immunogen. A lower but still measurable IgG response
(1/10,000) to the murine R3 antibody was obtained when monkeys were
immunized with the chimaeric antibody, contrary to the results obtained
with the mutant Vh version (FIG. 6). With the mutant VH R3 antibody no
response was measurable after two immunizations, and a small response (1
/10,000) was measured after 4 immunizations.
EXAMPLE 10
Modification of the Variable Domain Sequences of IOR-T1 Murine Monoclonal
Antibody to Humanize the Predicted T-cell Antigenic Sequences
The variable region sequences of heavy and light chains of IOR-T1 were
analyzed for T-cell antigenic sequences.
In the variable domain of the heavy chain 3 segments were predicted, they
are:
1. FR1 between amino acids 2-21.
2. FR1, CDR1, FR2 between amino acids 29-43.
3. FR4, CDR3 between amino acids 97-111.
FIG. 7 shows a comparison with the most homologous human sequence and the
replacement proposed, which are 5 at the FR1, 2 at the FR2 and 2 at the
FR4.
The same procedure with the light chain (FIG. 8) rendered the following
T-cell antigenic segments:
1. FR3 between amino acids 60-65.
2. FR3, CDR3 between amino acids 79-90.
3. CDR3 between aminoacids 93-95A.
After the analysis we proposed 5 replacement in FR3 at positions: 60, 63,
83, 85 and 87.
EXAMPLE 11
Modification of the Variable Domain Sequences of IOR-CEA1 Murine Monoclonal
Antibody to Humanize the Predicted T-cell Antigenic Sequences
The variable region sequences of heavy and light chains of IOR-CEA1 were
analyzed for T-cell antigenic sequences.
In the variable domain of the heavy chain two segments were predicted, they
are:
1. FR1 between amino acids 1-16.
2. CDR3 and FR4 between residues 96-110.
FIG. 9 shows a comparison with the most homologous human sequence and the
replacements proposed, which are 7 at the FR1 and 2 at the FR4.
The same analysis with the light chain (FIG. 10) rendered the following
T-cell antigenic segments:
1. FR1 between amino acids 1-14.
2. CDR2-FR3 between amino acids 55-70.
3. FR3-CDR3-FR4 between residues 74-100.
After the analysis we proposed 4 replacements in FR1 at positions 9, 10, 11
and 13, 11 replacements in FR3 at positions 58, 60, 63, 70, 75, 76, 78,
81, 83, 85 and 87, and 1 replacement in FR4 at position 100.
EXAMPLE 12
Analysis of Amphipatic Segments in Variable Regions of Immunoglobulin
Families
The program AMPHI was included as a subroutine in a program written for
reading and processing the immunoglobulin sequences from the Kabat Data
Base. In processing the sequences the following rearrangements were made:
Undefined amino acids of type GLX (possible GLN or GLU) were defined as GLN
(both GLN and GLU have similar hydrophilicity indexes: -0.22 and -0.64
respectively).
Undefined amino acids of type ASX (possible ASN or ASP, with hydrophilicity
indexes of -0.60 and -0.77) were defined as ASN.
Other undefined amino acids (empty spaces or "strange" symbols in the
sequences were defined as XXX (unknown). The program AMPHI assigns a
hydrophilicity value of 0.0 to these amino acids.
Sequences with more than 5 unknown amino acids (XXX) were not included in
the analysis.
After this preliminary analysis each sequence was processed by the program
AMPHI and the results are presented in the form of tables for each
immunoglobulin family.
In tables I to VI the analysis for the six mouse heavy chain families is
shown. "Predominant amphipatic regions" (PAR) could be defined at those
present in more than 90% of the variable region sequences belonging to
each family. For example, comparing the framework one (FR1), a PAR could
be defined between the 11 and the 16 amino acid residues for the families
I and II, by contrary families III and IV have not amphipatic regions in
general from the first amino acid to the 30th. In families V and VI,
smaller PARs could be defined from 12-14 and 12-15 residues respectively.
Humanization of the PARs would reduce immunogenicity in patients. The
clustering of amphipatic regions in the immunoglobulin variable region
frameworks supports the universality of the proposed method, i.e. to
humanize these predicted T-cell epitopes by few point mutations.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 31
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GGAAGCTTAGACCGATGGGGCCTGTTGTTTTG32
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GGAAGCTTGAAGATGGATACAGTTGGTGCAGC32
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
AGGTSMARCTGCAGSAGTCWGG22
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
TTGAATTCCAGTGATGTTTTGATGACCCA29
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG34
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GACATTCAGCTGACCCA17
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GTTAGATCTCCAGTTTGGTGCT22
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GAAGCCCCAGGCTTCTTCACTTCAGCCCCAGGCTG35
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GTAAAACGACGGCCAGT17
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CAGCCTGGGGCTGAAGTGAAGAAGCCTGGGGCTTCA36
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ACTGGCCGTCGTTTTAC17
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GCAGAGTCCTCAGATCTCAGGCTGCTGAGTTGCATGTAGACTGTGCTGGTGGATTCGTCT60
ACCGT65
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ACGGTAGACGAATCCACCAGCACAGTCTACATGCAACTCAGCAGCCTGAGATCTGAGGAC60
TCTGC65
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
AspValLeuMetThrGlnIleProLeuSerLeuProValSerLeuGly
151015
AspGlnAlaSerIleSerCysArgSerSerGlnAsnIleAsnIleVal
202530
HisSerAsnGlyAsnThrTyrLeuAspTrpTyrLeuGlnLysProGly
354045
GlnSerProAsnLeuLeuIleTyrLysValSerAsnArgPheSerGly
505560
ValProAspArgPheArgGlySerGlySerGlyThrAspPheThrLeu
65707580
LysIleSerArgValGluAlaGluAspLeuGlyValTyrTyrCysPhe
859095
GlnTyrSerHisValProTrpThrPheGlyGlyGlyThrLysLeuGlu
100105110
IleLysArgAla
115
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GlnValGlnLeuGlnGlnProGlyAlaGluLeuValLysProGlyAla
151015
SerValLysLeuSerCysLysAlaSerGlyTyrThrPheThrAsnTyr
202530
TyrIleTyrTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIle
354045
GlyGlyIleAsnProThrSerGlyGlySerAsnPheAsnGluLysPhe
505560
LysThrLysAlaThrLeuThrValAspGluSerSerThrThrAlaTyr
65707580
MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCys
859095
ThrArgGlnGlyLeuTrpPheAspSerAspGlyArgGlyPheAspPhe
100105110
TrpGlyGlnGlyThrThrLeuThrValSerSer
115120
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla
151015
SerValLysValSerCysLysAlaSerGlyTyrThrPheAsnTrpVal
202530
ArgGlnAlaProGlyGlnGlyLeuGluTrpMetGlyArgValThrMet
354045
ThrArgAspThrSerThrSerThrValTyrMetGluLeuSerSerLeu
505560
ArgSerGluAspThrAlaValTyrTyrCysAlaArgTrpGlyGlnGly
65707580
ThrLeuValThrValSerSer
85
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GlnValGlnLeuGlnGlnProGlyAlaGluValLysLysProGlyAla
151015
SerValLysLeuSerCysLysAlaSerGlyTyrThrPheThrAsnTyr
202530
TyrIleTyrTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIle
354045
GlyGlyIleAsnProThrSerGlyGlySerAsnPheAsnGluLysPhe
505560
LysThrLysAlaThrLeuThrValAspGluSerThrSerThrValTyr
65707580
MetGlnLeuSerSerLeuArgSerGluAspSerAlaValTyrTyrCys
859095
ThrArgGlnGlyLeuTrpPheAspSerAspGlyArgGlyPheAspPhe
100105110
TrpGlyGlnGlyThrThrLeuThrValSerSer
115120
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
AspValLeuMetThrGlnIleProLeuSerLeuProValSerLeuGly
151015
AspGlnAlaSerIleSerCysArgSerSerGlnAsnIleValHisSer
202530
AsnGlyAsnThrTyrLeuAspTrpTyrLeuGlnLysProGlyGlnSer
354045
ProAsnLeuLeuIleTyrLysValSerAsnArgPheSerGlyValPro
505560
AspArgPheArgGlySerGlySerGlyThrAspPheThrLeuLysIle
65707580
SerArgValGluAlaGluAspLeuGlyValTyrTyrCysPheGlnTyr
859095
SerHisValProTrpThrPheGlyGlyGlyThrLysLeuGluIleLys
100105110
Arg
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 81 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
AspValValMetThrGlnSerProLeuSerLeuProValThrLeuGly
151015
GlnProAlaSerIleSerCysTrpPheGlnGlnArgProGlyGlnSer
202530
ProArgArgLeuIleTyrGlyValProAspArgPheArgGlySerGly
354045
SerGlyThrAspPheThrLeuLysIleSerArgValGluAlaGluAsp
505560
ValGlyValTyrTyrCysPheGlyGlnGlyThrLysValGluIleLys
65707580
Arg
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
GluValLysLeuValGlnSerGlyGlyGlyLeuValLysProGlyGly
151015
SerLeuLysLeuSerCysAlaAlaSerGlyPheLysPheSerArgTyr
202530
AlaMetSerTrpValArgGlnThrProGluLysArgLeuGluTrpVal
354045
AlaThrIleSerSerGlyGlySerSerHisLeuLeuSerArgGlnCys
505560
GluGlyArgPheThrIleSerArgAspAsnValLysAsnThrLeuTyr
65707580
LeuGlnMetSerSerLeuArgSerGluAspThrAlaMetTyrTyrCys
859095
AlaArgArgAspTyrAspLeuAspTyrPheAlaSerTrpGlyGlnGly
100105110
ThrThrLeuThrValSerSer
115
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly
151015
SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerTrpVal
202530
ArgGlnAlaProGlyLysGlyLeuGluTrpValSerArgPheThrIle
354045
SerArgAspAsnSerLysAsnThrLeuTyrLeuGlnMetAsnSerLeu
505560
ArgAlaGluAspThrAlaValTyrTyrCysAlaLysTrpGlyGlnGly
65707580
ThrLeuValThrValSerSer
85
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly
151015
SerLeuArgLeuSerCysAlaAlaSerGlyPheLysPheSerArgTyr
202530
AlaMetSerTrpValArgGlnAlaProGlyLysArgLeuGluTrpVal
354045
SerThrIleSerSerGlyGlySerSerHisLeuLeuSerArgGlnCys
505560
GluGlyArgPheThrIleSerArgAspAsnValLysAsnThrLeuTyr
65707580
LeuGlnMetSerSerLeuArgSerGluAspThrAlaMetTyrTyrCys
859095
AlaArgArgAspTyrAspLeuAspTyrPheAlaSerTrpGlyGlnGly
100105110
ThrLeuValThrValSerSer
115
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
AspIleValMetThrGlnAspGlnLysPheMetSerThrSerValGly
151015
AspArgValSerValThrCysLysAlaSerGlnAsnAlaGlyThrAsn
202530
ValAlaTrpTyrGlnGlnLysProGlyGlnSerProLysAlaLeuIle
354045
TyrSerAlaSerSerArgAsnSerGlyValProAspArgPheThrGly
505560
SerGlySerGlyThrAspPheThrLeuThrIleSerAsnValGlnSer
65707580
GluAspLeuAlaGluTyrPheCysGlnGlnTyrAsnSerTyrProLeu
859095
ValThrPheGlyAlaGlyThrLysLeuGluLeuLysArgAla
100105110
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GluIleValMetThrGlnSerProAlaThrLeuSerValSerProGly
151015
GluArgAlaThrLeuSerCysTrpTyrGlnGlnLysProGlyGlnPro
202530
ProArgLeuLeuIleTyrGlyIleProAlaArgPheSerGlySerGly
354045
SerGlyThrGluPheThrLeuThrIleSerArgLeuGlnSerGluAsp
505560
PheAlaValTyrTyrCysPheGlyGlnGlyThrArgValGluIleLys
65707580
ArgGlu
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
AspIleValMetThrGlnAspGlnLysPheMetSerThrSerValGly
151015
AspArgValSerValThrCysLysAlaSerGlnAsnAlaGlyThrAsn
202530
ValAlaTrpTyrGlnGlnLysProGlyGlnSerProLysAlaLeuIle
354045
TyrSerAlaSerSerArgAsnSerGlyValProAlaArgPheSerGly
505560
SerGlySerGlyThrAspPheThrLeuThrIleSerAsnValGlnSer
65707580
GluAspPheAlaValTyrTyrCysGlnGlnTyrAsnSerTyrProLeu
859095
ValThrPheGlyAlaGlyThrLysLeuGluLeuLysArgAla
100105110
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 120 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GlnProLysLeuLeuGluSerGlyGlyAspLeuValLysProGluAla
151015
SerLeuAsnCysSerCysAlaValSerGlyPheProPheAsnArgTyr
202530
AlaMetSerTrpValLeuGlnThrProGluLysArgLeuGluTrpVal
354045
AlaPheIleSerSerAspAspGlyIleAlaTyrTyrAlaGluSerLys
505560
GlyTyrArgPheThrIleSerArgAspAsnAlaLysAsnIleLeuTyr
65707580
LeuGlnMetSerSerLeuArgSerGlnAspThrAlaMetTyrTyrCys
859095
AlaArgValTyrTyrTyrGlySerSerTyrPheAspTyrTrpGlyGln
100105110
GlyThrThrLeuThrValSerSer
115120
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla
151015
SerLeuLysValSerCysLysAlaSerGlyTyrPheThrTrpValArg
202530
GlnAlaProGlyGlnArgLeuGluTrpMetGlyArgValThrIleThr
354045
ArgAspThrSerAlaSerThrAlaTyrMetGluLeuSerSerLeuArg
505560
SerGluAspThrAlaValTyrTyrCysAlaArgTrpGlyGluGlyThr
65707580
LeuValThrValSerSer
85
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 120 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
GlnValGlnLeuValGlnSerGlyAlaGluLeuValLysProGlyAla
151015
SerLeuAsnCysSerCysAlaValSerGlyPheProPheAsnArgTyr
202530
AlaMetSerTrpValLeuGlnThrProGluLysArgLeuGluTrpVal
354045
AlaPheIleSerSerAspAspGlyIleAlaTyrTyrAlaGluSerLys
505560
GlyTyrArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr
65707580
LeuGlnMetSerSerLeuArgSerGlnAspThrAlaValTyrTyrCys
859095
AlaArgValTyrTyrTyrGlySerSerTyrPheAspTyrTrpGlyGln
100105110
GlyThrLeuValThrValSerSer
115120
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
AspIleGlnMetThrGlnSerProLysPheSerSerThrSerValGly
151015
AspArgValSerValThrCysLysAlaSerGlnAsnAlaGlyIleAsn
202530
ValAlaTrpTyrGlnGlnLysProGlyGlnSerProLysAlaLeuIle
354045
TyrSerAlaSerSerArgAsnSerGlyValProAspArgPheThrGly
505560
SerGlySerGlyThrAspPheThrLeuThrIleSerAsnValGlnSer
65707580
GlnAspLeuAlaGluTyrPheCysGlnGlnTyrAsnSerTyrProLeu
859095
ValThrPheGlyAlaGlyThrLysLeuGlnLeuLysArgThr
100105110
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
AspIleGlnMetThrGlnSerProSerThrLeuSerAlaSerValGly
151015
AspSerIleThrIleThrCysTrpPheGlnGlnLysProGlyLysAla
202530
ProAsnValLeuIleTyrGlyIleProSerArgPheSerGlySerGly
354045
SerGlyThrGluPheThrLeuThrValIleAsnLeuGlnSerAspAsp
505560
PheAlaThrTyrTyrCysPheGlyGlnGlyThrLysValLeuIleLys
65707580
ArgThr
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
AspIleGlnMetThrGlnSerProSerThrLeuSerAlaSerValGly
151015
AspArgValSerValThrCysLysAlaSerGlnAsnAlaGlyIleAsn
202530
ValAlaTrpTyrGlnGlnLysProGlyGlnSerProLysAlaLeuIle
354045
TyrSerAlaSerSerArgAsnSerGlyIleProSerArgPheSerGly
505560
SerGlySerGlyThrGluPheThrLeuThrValIleAsnLeuGlnSer
65707580
AspAspPheAlaThrTyrTyrCysGlnGlnTyrAsnSerTyrProLeu
859095
ValThrPheGlyGlnGlyThrLysLeuGlnLeuLysArgThr
100105110
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